The tumor suppressor p53 is a sequence-specific transcription factor that regulates an extensive network of coding genes, long non-coding RNAs and microRNAs, that establish intricate gene regulatory circuits influencing many cellular responses beyond the prototypical control of cell cycle, apoptosis and DNA repair.
Bisio et al BMC Cancer 2013, 13:552 http://www.biomedcentral.com/1471-2407/13/552 RESEARCH ARTICLE Open Access Identification of new p53 target microRNAs by bioinformatics and functional analysis Alessandra Bisio1, Veronica De Sanctis1,4, Valerio Del Vescovo2, Michela A Denti2, Anil G Jegga3, Alberto Inga1 and Yari Ciribilli1* Abstract Background: The tumor suppressor p53 is a sequence-specific transcription factor that regulates an extensive network of coding genes, long non-coding RNAs and microRNAs, that establish intricate gene regulatory circuits influencing many cellular responses beyond the prototypical control of cell cycle, apoptosis and DNA repair Methods: Using bioinformatic approaches, we identified an additional group of candidate microRNAs (miRs) under direct p53 transcriptional control To validate p53 family-mediated responsiveness of the newly predicted target miRs we first evaluated the potential for wild type p53, p63β and p73β to transactivate from p53 response elements (REs) mapped in the miR promoters, using an established yeast-based assay Results: The REs found in miR-10b, -23b, -106a, -151a, -191, -198, -202, -221, -320, -1204, -1206 promoters were responsive to p53 and of them were also responsive to p63β or p73β The potential for germline p53 mutations to drive transactivation at selected miR-associated REs was also examined Chromatin Immuno-Precipitation (ChIP) assays conducted in doxorubicintreated MCF7 cells and HCT116 p53+/+ revealed moderate induction of p53 occupancy at the miR-202, -1204, -1206, -10b RE-containing sites, while weak occupancy was observed for the miR-23b-associated RE only in MCF7 cells RT-qPCR analyses cells showed modest doxorubicin- and/or Nutlin-dependent induction of the levels of mature miR-10b, -23b, -151a in HCT116 p53+/+ and MCF7 cells The long noncoding RNA PVT1 comprising miR-1204 and −1206 was weakly induced only in HCT116 p53+/+ cells, but the mature miRs were not detected miR-202 expression was not influenced by p53-activating stimuli in our cell systems Conclusions: Our study reveals additional miRs, particularly miR-10b and miR-151a, that could be directly regulated by the p53-family of transcription factors and contribute to the tuning of p53-induced responses Keywords: p53 family, miRNA, Promoter occupancy, Transcription regulation Background The master regulator p53 is a prominent tumor suppressor gene, functioning in the cell as a tetrameric (dimer of dimers) sequence-specific transcription factor, able to bind to two copies of a decameric sequence with the RRRCWWGYYY consensus (where R stands for a purine, W for A/T and Y for a pyrimidine) representing the so called p53-response element (p53-RE) [1] p53 is known to be inducible in response to a large number of cellular stress signals that, besides genotoxic stress, include carbon * Correspondence: ciribilli@science.unitn.it Laboratory of Transcriptional Networks, Center for Integrative Biology, CIBIO, University of Trento, Trento, Italy Full list of author information is available at the end of the article and oxygen deficiencies, perturbations of the translation apparatus, excessive proliferation signals, alteration in microtubule dynamics [2,3] There are >100 established p53 targets genes that link p53 to cell cycle arrest, apoptosis, DNA repair and inhibition of angiogenesis [3-6] More recently, p53 was demonstrated to modulate the expression of genes able to modify glucose as well as lipid metabolism, induction of autophagy, immune responses and cell motility [7-11] A direct role of p53 on the activation of microRNA (miRs) expression as well as a role on selective maturation of microRNA precursors has been recently established [12,13] miRs are small non coding RNAs typically of 21– 25 nucleotides in length that regulate gene expression by © 2013 Bisio et al.; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Bisio et al BMC Cancer 2013, 13:552 http://www.biomedcentral.com/1471-2407/13/552 inhibiting translation or repressing stability of target messenger RNAs including those coding for oncogenes and tumor suppressor proteins [14] Dysregulation in miR expression has been reported in various cancers and can contribute to tumorigenesis [15] The first evidence of a p53-dependent regulation of miR genes was provided by He et al., who identified a family of miRs, namely miR34a-c, whose expression reflected the p53 status [16] The authors demonstrated that genes encoding miR-34 family cluster were direct transcriptional targets of p53 and that their induced expression levels upon genotoxic or oncogenic stress was dependent on p53 expression, both in vitro and in vivo Moreover, He et al identified the DNA sequences responsible for the p53 responsiveness of those miRs A year later another group of miRs, (miR-192, -194 and −215) was identified as targets of p53 and their ability to increase the level of CDKN1A (p21CIP1) and to function as drivers of cell cycle arrest was established [17] Examples of feedback loops or regulatory circuits comprising p53, a target miR and target mRNAs were discovered For example, p53-directed repression of c-Myc has also been linked to p53-dependent induction of miR145 [18] miR-107 was demonstrated to be activated by p53 and to cooperate in its cancer suppressive function through the inhibition of HIF-1β and, consequently, tumor angiogenesis [19] The p53 targeted miR-34a was shown to modulate SIRT1 [20] More recently, Jin et al surprisingly found that p53 directly induced the transcription of miR149*, which in turn can target the glycogen synthase kinase-3α mRNA, resulting in elevated expression of Mcl-1 and resistance to apoptosis in melanoma cells, thus providing a rational explanation for the poor ability of p53 to suppress melanoma progression [21] Furthermore, it has been demonstrated that p53 itself can be indirectly activated by the miR-29 family members (miR-29a, -29b and -29c), which inhibit the expression of p85 alpha (the regulatory subunit of the phosphatidylinositol-3 kinase, PI3K) and CDC42 (Cell division cycle 42, a Rho family GTPase), thereby decreasing their inhibitory effect on p53 [22] Alternatively, miRs can also negatively regulate p53 expression as observed for miR-1285, miR-504, miR-33, miR-380, miR-30d, miR-25 and miR-125b [23-28] The mechanisms regulating in vivo p53 transactivation specificity still need to be fully understood, but require in most cases the interaction of p53 with its response element sequences (REs) at target promoters [4] Recent evidences, including our studies using functional as well as DNA binding assays in yeast or mammalian cells or with cell extracts, demonstrated that maximal transactivation potential requires adjacent dimer binding sites [29-34] A spacer between dimer sites even of or nucleotides conferred a negative impact, particularly for the p53-related protein p73 [31,35,36] We also established that p53 can Page of 13 stimulate transcription, albeit at a reduced levels, from noncanonical response elements (half-sites and ¾ sites) [31], that not provide for a p53 tetramer binding site The same sequence-specific requirements that were shown to maximize the transactivation potential from full-site REs, appeared to be valid for the half-site REs [31] This information is relevant to optimize pattern-based motif searches aiming at identifying functional p53 response elements within genomes [37-39] In this study we used a regression-based predictor for p53 transactivation [39], to identify additional p53target miRs through the presence of functional p53-REs in their promoter regions or in promoter regions of long noncoding RNA that are precursors of those miRs We then used a yeast-based functional assay to determine the relative transactivation capacity of p53 family proteins towards the identified REs and Chromatin ImmunoPrecipitation (ChIP) assays in human cells to investigate genotoxic-stress dependent p53 occupancy at the chromosomal sites containing those REs Changes in the expression levels for mature miRs or precursors were measured by real-time qPCR using cell lines and treatments probing the direct involvement of p53 We propose miR-10b, -23b and -151a to be included in the list of direct p53 target miRs contributing to the fine-tuning of p53-induced responses Methods Yeast reporter strains and media We constructed a panel of 16 reporter strains in the budding yeast Saccharomyces cerevisiae containing the Firefly luciferase gene under the control of putative p53-REs (see Table for the RE sequence and location with respect to miR genomic coordinates) predicted to control the expression of miR -10b, -23b, -34a, -106a, -145, -151a, -191, -198, -202, -221, -320, -328, -455, -671, -1204, -1206 To this aim we took advantage of the methodology of the well-established delitto perfetto approach for in vivo mutagenesis using oligonucleotides [40] starting with the master reporter strain yLFM-ICORE [41] The strain contains the luciferase cDNA integrated at the chromosome XV downstream a minimal promoter derived from the CYC1 gene The ICORE cassette is located 5′ to the minimal promoter and enables high efficiency targeting of the locus by oligonucleotides that contain desired RE sequences The targeting events were followed by phenotypic selection and clones examined by colony PCR and direct DNA sequencing (BMR Genomics, Padua, Italy) Yeast cells were grown in 1% yeast extract, 2% peptone, 2% dextrose with the addition of 200 mg/L adenine (YPDA medium) Selective minimal plates lacking tryptophan or leucine but containing adenine (200 mg/L) and dextrose as carbon sources were used (transformation in yeast and luciferase-based assay) Bisio et al BMC Cancer 2013, 13:552 http://www.biomedcentral.com/1471-2407/13/552 Page of 13 Table List of predicted p53-REs mapped nearby miR genes miR name p53-RE Genomic location p53-RE distance miRNA Overlapping transcript Distance from sense transcript TSS - - - miR-34a Chr - 9242203 - 30,476 bp miR-10b Chr - 177013750 - 1,281 bp Consensus p53- RE organization DIMER Spacer DIMER - RRRCATGYYY N = - 13 RRRCATGYYY Intergenic + 123 bp GGGCTTGCCT - GGGCTTGTTC HOXD3 + 12,065 bp TAACTCGTTG GCTTTGACCTGTCT GAACAAGTCG miR-23b Chr - 97818687 - 28,803 bp C9orf3 + 135 bp AGGTCAGTCA TG GGACATGTCC miR-106a Chr X - 133309906 - 5,678 bp Intergenic - GTTATGTTC ATGTGCTCAT GTGCATGCCC miR-145 Chr - 148786372 - 23,837 bp Intergenic + 68 bp GCACCCGCCT CGCCCCAATACG GGGCTTGCCT miR-151a Chr - 141734774 - 2,349 bp PTK2 - 75,874 bp TGGCTTGTTT - TGGCAAGTTC miR-191 Chr - 49063594 - 5,543 bp DARLD3 + 5,103 bp GACCTTGTCT TGCTTCC AGCCATGTCA miR-198 Chr - 120112741 + 1,774 bp FSTL1 - 57,359 bp AGGCAAGCTT - CAACAAGCCG miR-202 Chr 10 - 135058647 + 2,368 bp RP13-49115.5 - 2,748 bp GGGCATGTCT - TGGCAAGCCT miR-221 Chr X - 45599946 + 5,639 bp Intergenic - GAACATGCAT - GCACATGTTT miR-455 Chr - 116880600 - 91,114 bp COL27A1 - 49,395 bp CTTCCTGCAT AAGGCTTGGCGG GCGCAAGCCC miR-320a Chr - 22095461 + 7,014 bp Intergenic - AGGCATGGTG - CGGCATGCCT miR-328 Chr 16 - 67331546 - 95,322 bp ELMO3 + 98,475 bp CGGCAAGTCC C CAGCCAGTTC miR-671 Chr - 150896632 - 38,875 bp CHPF2 - 32,953 bp GGTCCAGCCC TCTGGCCCCC CAACAAGTCT miR-1204 Chr - 128808017 - 191 bp PVT1 + 1,238 bp CGACAAGTTG - AGACTTGTTC miR-1206 Chr - 129002467 - 18,677 bp PVT1 + 1,037 bp GGGCTAGTGA - AGGCATGTCT miR name, genomic location of the predicted p53-RE (including chromosome number and genomic coordinates according to BLAT tool in UCSC Genome Browser, http://genome.ucsc.edu/), presence and distance from an overlapping primary transcript, p53 RE sequence and position relative to the miR sequence (obtained through the miRBase tool, http://www.mirbase.org/) are presented p53-RE organization is shown grouping nucleotides in Dimer 1, Spacer (if any) and Dimer 2, according to the p53 consensus (in bold) Yeast expression vectors For the expression of p53 family protein in yeast we used available CEN/ARS expression vectors (pTSG- or pLSGbased) harbouring alternatively the cDNA wild-type of p53, p63 or p73 under the control of the GAL1,10 inducible promoter [31] Among the various isoforms of p53 family members, we selected the full-length wild type p53 and the TA-p63β and TA-p73β isoforms as they showed the maximal transactivation potential in our experimental settings The expression levels were modulated by the concentration of galactose in the culture medium (0.008%, 0.128% or 1%) The whole panel of 104 p53 germline missense mutants from the IARC R11 database (http://p53 iarc.fr) cloned in the pLS-Ad vector (providing for a constitutive expression of p53 mutants) [42] were used to test transactivation capability towards the miR-34a p53 RE The pRS-314 or pRS-315 empty vectors were included as controls; these vectors contain respectively the TRP1 (as pTSG-) or LEU2 (as pLSG-) yeast selectable markers Luciferase assays in yeast The p53 family responsiveness of miRNA-associated REs was examined by transforming the panel of yLFM-RE strains with the p53 expression vectors Transformants were obtained using the LiAc method and selected on minimal plates lacking tryptophan or leucine but containing adenine (200 mg/L) and dextrose as carbon source to allow respectively the growth of white colonies of normal shape and to keep the expression of p53 family members inhibited After days of growth at 30°C, transformants were streaked onto the same plates and allowed to grow for an additional day For each reporter strain, the basal luciferase activity was measured from pRS314- or pRS315transformants and used to calculate the fold of induction due to the expression of each p53 family member For the luciferase assay we exploited the recently developed miniaturized yeast assay [43] Transformant colonies were grown in 100 μl of the selective medium with 2% raffinose as carbon source (raffinose result in basal level of expression from the GAL1,10 promoter), supplemented with different concentrations of galactose (0.008% and 0.128% for p53; 0.008% and 1% for the other members of the family, to obtain a low, moderate or high expression of the proteins) in a transparent 96-well plate and incubated under 150 rpm shaking for 16–24 hours at 30°C OD600 was measured directly in the multi-well plate to normalize for cell density using a multilabel plate reader (Infinite M200Pro, Tecan, Milan, Italy) 10 μl of cells suspensions were transferred to a white 384-well plate (Brand, Milan Italy) and mixed with an equal volume of PLB buffer 2X (Passive Lysis Buffer, Promega, Milan, Italy) After 15 minutes of shaking at room temperature, 10 μl of Firefly luciferase substrate (Bright Glo Luciferase Reporter Assay, Promega) were added Luciferase activity was measured at Bisio et al BMC Cancer 2013, 13:552 http://www.biomedcentral.com/1471-2407/13/552 the plate reader, using a luminescence program with 1” integration time Cell lines and treatments The human breast adenocarcinoma-derived MCF7 cell line was obtained from the InterLab Cell Line Collection bank, ICLC (Genoa, Italy) The colon adenocarcinoma HCT116 (p53+/+) cell line and its p53−/− derivative were obtained from B Vogelstein’s group (The Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland, USA) Cells were normally maintained in DMEM or RPMI supplemented with 10% FCS and antibiotics (100 units/ml penicillin plus 100 mg/ml streptomycin) Cells were treated for 24 hours with 1.5 μM doxorubicin (DXR) or 10 μM Nutlin-3A for p53 stabilization/activation Stock solutions of Nutlin-3A were dissolved in 100% DMSO (stock solution 10 mM); DXR was dissolved in H2O (stock 10 mM) DXR was bought from SigmaAldrich® (Milan, Italy) whereas Nutlin-3A from Alexis® Biochemicals (Enzo Life Sciences, Exeter, UK) All the treatments were performed when cells reached 70-80% of confluence RNA extraction and RT-qPCR RNA extraction from human cell lines was done with Trizol reagent (InVitrogen, Life Technologies, Milan, Italy) following the manufacturer’s protocol Cells were treated with 1.5 μM DXR or 10 μM Nutlin-3A for 24 hours prior to Trizol extraction Quantification of mature microRNA expression was carried out using TaqMan MicroRNA Assay kits according to the manufacturer’s protocol (Applied Biosystems, Life Technologies), as described in Barbareschi et al [44] Specifically, ready-made TaqMan MicroRNA Assays (containing microRNA-specific forward and reverse PCR primers and microRNA-specific Taqman MGB probe) were used for the investigation of miR-34a (ABI P/N 000426), miR-10b (ABI P/N 000388), miR-23b (ABI P/N 000400), miR-151a (ABI P/N 002254), and miR-202 (ABI P/N 002363) We also quantified the U6 small nuclear RNA (RNU6B) (ABI P/N 4373381) as an endogenous control to normalize the levels of target microRNA Complementary DNA was generated using the Taqman MicroRNA Reverse Transcription (RT) Kit (ABI P/N 4366596) according to the manufacturer’s instructions Reverse transcriptase reactions contained 10 ng of total RNA as the template, μl of gene specific stemloop RT primer, 1.5 μl of 10X RT buffer, 0.15 μl of 100 mM dNTPs, μl of MultiScribe reverse transcriptase, and nuclease-free water The 15 μl reactions were incubated on a GeneAmp PCR System (Bio-Rad, Hercules, CA) for 30 minutes at 16°C, 30 minutes at 42°C, minutes at 85°C, and then kept at 4°C Quantitative RT-PCR was carried out using the Rotorgene 6000 (Corbett Life Science, Ancona, Italy) in 20 μl PCR reactions containing Page of 13 1.33 μl of RT product, 10 μl of FastStart TaqManProbe Master (Roche, Milan, Italy, P/N04673417001), 7.67 μl of nuclease–free water, and μl of MicroRNA Assay (Applied Biosystems, Life Technologies) buffer Reactions were incubated at 95°C for 10 minutes, followed by 40 cycles of incubation at 95°C for 15 seconds and at 60°C for minute The quantification of protein coding mRNAs was performed using a Sybr green RT-qPCR approach Total RNAs extracted with Trizol were converted using the RevertAid™ First Strand cDNA Synthesis Kit containing the M-MuLV Reverse Transcriptase following the manufacture’s recommendation (Thermo Scientific Inc., M-Medical, Milan, Italy) qPCR were carried out using the KAPA Sybr Green PCR mix (Kapa Biosystems, Resnova, Rome, Italy) with 12.5 ng of cDNA on the CFX384 real-time PCR detection system (BioRad, Milan, Italy) Primers were picked using the Primer-BLAST online tool (http://www.ncbi.nlm nih.gov/tools/primer-blast/); sequences are available upon request The Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β2-microgobulin (B2M) were used as reference genes for normalization In all the qPCR assays, the threshold cycle data (Ct) and baselines were determined using auto settings The Ct value was defined as the fractional cycle number at which the fluorescence passed a fixed threshold Fold changes were calculated using the comparative Ct method (ΔΔCt) Western blot analysis To evaluate p53, p63 or p73 protein levels in yeast we cultured transformant colonies for 24 hours using selective medium containing 0.128% (p53) or 1% (p63β and p73β) galactose to induce the expression Yeast cells were harvested, washed in ddH2O and lysed mechanically with glass beads as previously described [45] 15 μg (p53) and 75 μg (p63β and p73β) were loaded on a 7.5% Acrylamide gel and separated by SDS-PAGE DO-1, 4A4 (Santa Cruz Biotechnology, Milan, Italy) and ER-15 (Calbiochem, Merck, Millipore, Milan, Italy) antibodies were used for p53, p63 and p73 immunodetection, respectively PhosphoGlycerate Kinase (PGK1, detected by Ab clone number 22C5D8, InVitrogen, Life Technologies) was used as loading control To demonstrate p53 stabilization and activation upon treatment with doxorubicin or Nutlin-3A, MCF7, HCT p53+/+ and HCT p53−/− cells were harvested 16–18 hours after the treatments and lysed using RIPA (Radio Immuno Precipitation Assay) buffer (150 mM sodium chloride; 1.0% NP-40; 0.5% sodium deoxycholate; 0.1% SDS, sodium dodecyl sulphate; 50 mM Tris, pH 8.0) supplemented with Protease Inhibitors cocktail (Roche, Milan, Italy) [46] 50 μg of the soluble extracts were loaded on a 12% Acrylamide gel and separated by SDS-PAGE p53 and p21 endogenous protein levels were detected with incubation with monoclonal antibodies (DO-1 and C-19 clones against p53 and p21 Bisio et al BMC Cancer 2013, 13:552 http://www.biomedcentral.com/1471-2407/13/552 respectively, Santa Cruz Biotechnology) Glyceraldehyde 3phosphate dehydrogenase protein (GAPDH, Ab clone 6C5, Santa Cruz Biotechnology) served as loading control All antibodies were diluted in 1% non-fat skim milk dissolved in PBS-0.1% Tween20 Chromatin immunoprecipitation (ChIP) analysis HCT116 p53+/+ and HCT116 p53−/− or MCF7 cells were grown on 150-mm dishes and treated with 1.5 μM doxorubicin for 24 hours Proteins were cross-linked with DNA by addition of 1% formaldehyde After 10 minutes incubation at room temperature the reaction was stopped by addition of glycine at a final concentration of 0.125 M followed by additional incubation for minutes Cells were washed twice with 10 ml cold PBS, harvested in ml PBS plus protease inhibitors (PI) (Complete EDTA-free, Roche), and lysed using an SDS lysis buffer (1% SDS, 50 mM Tris–HCl pH 8, 20 mM EDTA, 0.1 mg/mL Sonicated Salmon sperm DNA, 1X PI) In order to eliminate soluble p53 protein, lysates were incubated with gently shaking for 10 and insoluble material was collected by centrifugation at 800 g at 4°C for Pellets were resuspended in 0.5 ml of sonication buffer containing 0.25% SDS, 200 mM NaCl, 100 mg/ml of sonicated salmon sperm DNA and protease inhibitors and were sonicated to shear DNA to lengths between 150 and 500 base pairs (bp) using a Misonix S-4000 sonicator with a plate horn (Misonix, Newtown, Connecticut, USA) After 10-fold dilution in ChIP dilution buffer (16.7 mM Tris, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl), IPs were carried out overnight at 4°C with μg of anti-p53 (DO-1, Santa Cruz Biotechnology) or μg of normal IgG as a negative control Fifty microliters of Dynabeads protein G magnetic beads (Invitrogen, Life Technologies) were added to each sample for 2–3 h, and the beads were then washed as indicated in the Upstate Biotechnology ChIP protocol DNA was eluted firstly with 100 μL of TE with 1% SDS for 10 at 65°C and a second time with 150 μL of TE with 0.67% SDS for an additional 10 at 65°C The cross-links were reversed overnight at 65°C RNase A was added and incubated at 37°C for 30 and then Proteinase K for h at 56°C DNA was then purified by QIAquick PCR purification KIT columns (Qiagen, Milan, Italy) Immunoprecipitated DNA was analyzed for p53 occupancy on selected chromosomal sites surrounding the predicted miR-associated p53 REs by RealTime-qPCR and fold enrichment of p53 binding to the respective DNA sequences was calculated by the comparative Ct method as described previously [47] RealTime-qPCR was carried out with the KAPA SYBR Green PCR mix (Kapa Biosystems, Resnova) and all primers were checked for equal amplification efficiency All PCR results were normalized to input controls Three different DNA loci were used as ChIP negative controls Page of 13 (the CCNB1 exon 9, the Actin and GAPDH promoter) Sequences of all ChIP primers are available upon request Results and discussion Identification of functional p53 response elements in miR genes We applied a predictor tool for p53 RE-transactivation potential [39] to identify candidate p53 REs within regulatory regions of miR genes or promoter regions of long noncoding RNAs containing pri-miR clusters Based on this analysis several novel p53 target miRs can be predicted (Table 1) To confirm p53 responsiveness of the identified p53 REs we first applied a well-established quantitative reporter assay in the budding yeast Saccharomyces cerevisiae [37,43] This assay was chosen as it provides a defined experimental system to measure transactivation potential of a panel of REs each cloned at the same chromosomal location in isogenic derivative reporter strains where wild type or mutant p53, as well as p53-related proteins p63 and p73, can be expressed, one at a time, from a inducible promoter [43] 15 candidate miR-associated p53 REs were studied The validated miR-34a p53 RE was included as a positive control The results indicated that most of the identified p53-REs could be transactivated at low to moderate levels by wild type p53 (Figure 1A) As expected the responsiveness was proportional to p53 expression levels (Figure 1A and B) Based on results obtained both with low and high p53 expression, REs from miR34a, -10b, -202, -1204 were highly responsive (HR), from miR-23b, -151a, -221, -320, -1206 were moderately-respon sive (MR), from -106a, -191, -198 were weakly responsive Putative REs from miR-145, -328, -455, -671 were not responsive to p53 in the yeast-based assay Next we tested p63 and p73 responsiveness of the same panel of REs, using the transactivation competent, TAp63β and TA-p73β isoforms for these proof-of-principle experiments, as they exhibit higher relative transactivation potential compared to N-terminal truncated ΔN- and also compared to TA-p63α and TA-p73α isoforms [37] As expected the transactivation potential of p63 β and p73β were much lower compared to p53 (Figure 1C and D) Only a subset of p53-responsive REs was active with p63 and p73 and included miR-34a, -202 and −1204 REs Furthermore, differences in relative transactivation potential were observed in the comparison of the three family members For example, miR-34a and −1204 were more responsive to p63 than to p73 Furthermore, we observed examples of selective lack of responsiveness: p73 towards miR-10b and −221 REs; p63 towards mir-151a To verify the protein expression of the three p53 family members in yeast after galactose induction (0.128% and 1% galactose for p53 or p63 and p73, respectively, to match the experimental conditions used for the luciferase assays) we performed Bisio et al BMC Cancer 2013, 13:552 http://www.biomedcentral.com/1471-2407/13/552 Page of 13 Figure p53 family members can transactivate p53-REs found in miR-associated promoter regions A-B) Transactivation potential of p53 protein tested on a panel of 15 putative p53-dependent miR-REs and miR-34a-RE (a positive control) using the yeast functional assay The expression of p53 was modulated by increasing concentrations of galactose in the culture medium (A, 0.008%: a moderate p53 expression; B, 0.128%: high p53 expression) C-D) The same panel of p53 miR-REs was tested using the other members of the p53 family p63 and p73 (gray and black bars respectively) The expression of p63β and p73β isoforms was induced using two different concentrations of galactose (C, 0.008%: moderate expression; D, 1%: maximal induction) Results are presented as fold of induction calculated over an empty expression vector Bars plot the averages and standard deviations of at least three independent biological repeats (E) Western Blot establishing galactose-dependent expression of p53, p63 or p73 in yeast PGK1 was used as reference Bisio et al BMC Cancer 2013, 13:552 http://www.biomedcentral.com/1471-2407/13/552 a western blot using antibodies specific for each transcription factor (Figure 1E) For REs, representative of strong, moderate, weak, and nearly-absent responsiveness to wild type p53, the functional assay was extended to five p53 missense germline mutations, of which three retain partial function (A138S, C141Y, R337C) and two are loss of function (A138P, R175H) [42] Similar results were obtained with the responsive REs, confirming the functional classification of the p53 mutants examined (Figure 2); for example, A138S was near-wild type, while R337C was weakly active (